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One-year-old `Concord' vines were fertigated with 0, 5, 10, 15, or 20 mm N in a modified Hoagland's solution for 8 weeks during summer. Half of the vines fertigated at each N concentration were sprayed with 3% foliar urea twice in late September while the rest served as controls. Four vines from each treatment combination were destructively sampled during dormancy to determine the levels and forms of N and carbohydrates. Nitrogen fertigation during the summer only slightly increased vine N concentration whereas foliar urea application in the fall significantly increased vine N concentration. In response to foliar urea application, concentrations of both free amino acid-N and protein-N increased, but the ratio of protein N to amino acid N decreased. Arginine was the most abundant amino acid in free amino acids and proteins, and its concentration was linearly correlated with vine N concentration. Concentrations of total non-structural carbohydrates (TNC) decreased slightly in response to N supply from fertigation. Foliar urea application in the fall significantly decreased TNC concentration at each N fertigation level. Starch, glucose and fructose decreased in response to foliar urea applications, but sucrose concentration remained unaffected. Approximately 60% of the carbon decrease in TNC caused by foliar urea application was recovered in proteins and free amino acids. We conclude that free amino acids account for a larger proportion of the N in vines sprayed with foliar urea, but proteins remain as the main form of N storage. In response to foliar urea application, part of the carbon from TNC is incorporated into proteins and free amino acids, leading to a decrease in the carbon stored in TNC and an increase in the carbon stored in proteins and free amino acids.

One-year-old `Concord' grapevines (Vitis labrusca L.) were fertigated twice weekly for 11 weeks with a complete nutrient solution containing 1, 10, 20, 50 or 100 μmol iron (Fe) from ferric ethylenediamine di (o-hydroxyphenylacetic) acid (Fe-EDDHA). Leaf total Fe content did not increase in response to Fe supply, however both “active” Fe (extracted with 2, 2'-dipyridyl) and chlorophyll (Chl) content increased as applied Fe increased. At the lowest active Fe level, leaf absorptance and maximum PSII efficiency (Fv/Fm) were slightly decreased, and non-photochemical quenching was significantly greater. PSII quantum efficiency decreased curvilinearly as active Fe content decreased. On a Chl basis, the xanthophyll cycle pool size, lutein, and beta-carotene increased curvilinearly as active Fe decreased, and neoxanthin increased at the lowest Fe level. Activities of antioxidant enzymes superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase followed a similar trend and increased under Fe deficiency, when expressed on a Chl basis. Antioxidant metabolites also increased in response to Fe limitation. On a Chl basis, ascorbate (AsA), dehydroascorbate (DAsA), reduced glutathione (GSH) and oxidized glutathione (GSSG) content was greater at the lowest active Fe levels. We did not find a difference in the ratio of AsA to DAsA or GSH to GSSG. In conclusion, both photoprotective mechanisms, xanthophyll cyle-dependent thermal dissipation and the ascorbate-glutatione antioxidant system, are enhanced in response to iron deficiency to cope with excess absorbed light.

Four-year-old `Gala'/M.26 trees were grown under low (2.5 mm), medium (12.5 mm), or high (25 mm) N supply with balanced nutrients in sand culture and the cropload was adjusted to 5 fruit/cm² trunk cross-sectional area at 10 mm king fruit. After harvesting, half of the trees in each N treatment were sprayed twice with 3% urea a week apart in late September. Before budbreak the following spring, four trees from each treatment combination were destructively sampled for reserve nitrogen and carbohydrate analysis. Foliar urea application significantly increased tree N concentration and concentrations of both free amino acids and proteins, but decreased the concentration of total nonstructural carbohydrates (TNC) at each soil N supply level. When the carbon in free amino acids and proteins are taken into account, trees sprayed with foliar urea had similar levels of total sum of carbon in TNC, free amino acids and proteins. On a whole tree basis, trees sprayed with foliar urea had more N and less TNC. During the second year of the experiment, all the trees received normal N supply. Trees sprayed with foliar urea the previous fall had a significantly larger total leaf area and higher fruit set, fruit number, and total yield than those unsprayed. We conclude that fruit set and early fruit development as well as vegetative growth in spring is mainly determined by reserve nitrogen, not by reserve carbohydrates. Conversion of a portion of TNC to amino acids and proteins leads to better growth and fruiting of apple trees.

Apple leaf ADP-glucose pyrophosphorylase was purified over 1400-fold to apparent homogeneity with a specific activity of 58.9 units per mg of protein. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolysis direction, however, high concentrations of PGA (>2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T_0.5 (temperature at which 50% of the enzyme activity is lost after 5 min of incubation) of 52 °C. Incubation with 2 mm PGA or 2 mm Pi increased T_0.5 to 68 °C. Incubation with 2 mm dithiothreitol (DTT) decreased T_0.5 to 42 °C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T_0.5 at 52 °C. DTT-induced decrease in thermal stability was accompanied by monomerization of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerization of the small subunits of the enzyme induced by DTT. These findings indicate that the binding of PGA may have dual functions in regulating apple leaf AGPase activity—activating the enzyme and rendering the enzyme with a conformation more stable to thermal inactivation.

Four-year-old `Gala'/M.26 trees were grown under low (2.5 mm), medium (12.5 mm), or high (25 mm) N supply with balanced nutrients in sand culture and the cropload was adjusted to 5 fruit/cm² trunk cross-sectional area at 10 mm king fruit. At about 100 days after bloom, exposed fruit on the south side of the canopy were chosen for monitoring chlorophyll fluorescence and fruit peel samples were taken for measuring xanthophyll cycle pigments, antioxidant enzymes, and metabolites. At noon, the efficiency of excitation transfer (Fv'/Fm') of the sun-exposed peel was higher in the low N treatment than in the medium or high N treatments. Photochemical quenching coefficient did not differ between fruits in different N treatments. The Photosystem II operating efficiency was higher in the peel of low N fruit compared with medium N or high N fruit. However, maximum quantum efficiency (Fv/Fm) of fruit peel after overnight dark adaptation was similar across the N treatments. The xanthophyll cycle pool size expressed on peel area basis was larger in the high N fruit than in the low N fruit. This corresponds well with the thermal dissipation capacity, as indicated by efficiency of excitation transfer. Over 95% of the xanthophyll cycle pool in the sun-exposed side was present in the form of zeaxanthin and antheraxanthin at noon regardless of N treatments. Activities of superoxide dismutase and all the antioxidant enzymes and metabolites in the ascorbate-glutathione cycle were higher in the high N fruit than in low N fruit. The results indicate that apple fruit with a good N status have a higher photoprotective capacity in terms of xanthophyll cycle-dependent thermal dissipation and detoxification of reactive oxygen species compared with low N fruit.

Apple maturity is often assessed by starch hydrolysis index, skin color, soluble solids, flesh firmness, and the rate of ethylene evolution. In red-fruited apple cultivars, the intensity and extent of coloration is an important consideration in determining the time of fruit harvest. Negative relationships have been found between tree nitrogen (N) status and fruit skin pigmentation, but how N affects flesh starch breakdown has not been examined in detail. The objective of this study was to determine how N supply affects flesh starch breakdown relative to skin color development. Seven-year-old ‘Gala’/M.26 trees were provided with four levels of N (8.8, 26.4, 52.7, and 105.4 g N per tree) in a modified Hoagland's solution. The effects of N supply on yield, fruit quality, and fruit maturation were evaluated. At harvest, fruit in the lowest N treatment was significantly smaller and had lower soluble solids but higher starch concentration, better color, and higher firmness than those grown at higher N supplies. Increasing N supply decreased both anthocyanin synthesis and chlorophyll degradation in fruit skin. Flesh starch concentration was higher at higher N supply at 38 days before harvest but was lower at higher N supply at harvest. Starch degradation was completed earlier during cold storage with increasing N supply. These results indicate that increasing N supply delays skin red color development but accelerates flesh starch degradation in ‘Gala’ apples. These differential effects of N supply should be taken into account when assessing fruit maturity for optimizing harvest time.

Cytosolic fructose-1,6-bisphosphatase (cytoFBPase) (EC 3.1.3.11) occupies a strategic site in sucrose synthesis and has been demonstrated to play a key role in carbon partitioning between sucrose and starch in non-sorbitol forming plants. In addition to sucrose and starch, Sorbitol is the primary photosynthetic end product in the leaves of many tree fruit species in the Rosaceae family. To understand the biochemical regulation of photosynthetic carbon partitioning between sorbitol, sucrose and starch in sorbitol synthesizing species, we purified cytoFB-Pase to apparent homogeneity from apple leaves. The enzyme was a homotetramer with a subunit mass of 37 kDa. It was highly specific for fructose-1,6-bisphosphate with a Km of 3.1 μm and a Vmax of 48 units/mg protein. Either Mg²⁺ or Mn²⁺ was required for its activity with a Km of 0.59 mm and 62 μM, respectively. Li⁺, Ca²⁺, Zn²⁺, Cu²⁺ and Hg²⁺ inhibited whereas Mn²⁺ enhanced the Mg²⁺-activated enzyme activity. Fructose-6-phosphate was found to be a mixed type inhibitor with a Ki of 0.47 mm. Fructose 2,6-bisphosphate (F2,6BP) competitively inhibited the enzyme activity and changed the substrate saturation curve from hyperbolic to sigmoidal. Adenosine monophosphate (AMP) was a non-competitive inhibitor for the enzyme. F2,6BP interacted with AMP to inhibit the enzyme in a synergistic way. Dihydroxyacetone phosphate did not have inhibitory effect on apple leaf cytosolic FBPase activity. Sorbitol increased the susceptibility of the enzyme to the inhibition by F1,6BP. The presence of sorbitol in the reaction mixture led to a reduction in the enzyme activity.

Plants of Fragaria chiloensis cv. RCP-37 were grown with their root system split between two separate containers. Water was withheld from one container of each pair (drought side), while the other was subirrigated. Control plants were subirrigated in both containers. Over several days, a drought-side leaf exhibited reductions in stomatal conductance (g_s) and transpiration rate (T), while a subirrigated side leaf showed no change in either parameter. However, foliar water relations components (water, osmotic, and pressure potential) did not differ between the two leaves. The leaf on the subirrigated side exhibited g_s, T, and water relations components similar to leaves on control plants. The abscisic acid (ABA) content of xylem exudate, collected from a stolon emerging from the axils of the measured leaves, was highest from the drought side and was negatively correlated to g_s and T at some sampling dates. A root-derived drought stress signal, perhaps ABA, although other factors cannot be discounted, was limited within the plant to the drought side, even though water relations components indicated that water from the subirrigated side was allocated to all parts of the plant.

To determine the cause of zonal chlorosis of `Honeycrisp' apple leaves, we compared CO₂ assimilation, carbohydrate metabolism, xanthophyll cycle and the antioxidant system between chlorotic leaves and normal leaves. Chlorotic leaves accumulated higher levels of non-structural carbohydrates, particularly starch, sorbitol, sucrose, and fructose at both dusk and predawn, and no difference was found in total non-structural carbohydrates between predawn and dusk. CO₂ assimilation and the key enzymes in the Calvin cycle, ribulose 1,5-bisphosphate carboxylase/oxygenase, NADP-glyceraldehyde-3-phosphate dehydrogenase, phosphoribulokinase, stromal fructose-1,6-bisphosphatase, and enzymes in starch and sorbitol synthesis, ADP-glucose pyrophosphorylase, cytosolic fructose-1,6-bisphosphatase, and aldose 6-phosphate reductase were significantly lower in chlorotic leaves than in normal leaves. However, sucrose phosphate synthase activity was higher in chlorotic leaves. Thermal dissipation of excitation energy was enhanced in chlorotic leaves under full sun, lowering the efficiency of excitation energy transfer to PSII reaction centers. This was accompanied by a corresponding increase in both xanthophyll cycle pool size (on a chlorophyll basis) and conversion of violaxanthin to antheraxanthin and zeaxanthin. The antioxidant system was up-regulated in chlorotic leaves in response to the increased generation of reactive oxygen species. These findings support the hypothesis that phloem loading and/or transport is partially or completely blocked in chlorotic leaves, and that excessive accumulation of non-structural carbohydrates may cause feedback suppression of CO₂ assimilation via direct interference with chloroplast function and/or indirect repression of photosynthetic enzymes.

About 80 days after full bloom, well-exposed fruit on the south part of the canopy of mature Liberty/M.9 apple trees were randomly assigned to one of the following two treatments. Some fruit were turned about 180 degrees to expose the original shaded side to full sun whereas the rest served as untreated controls. On day 0, 1, 2, 4, 7, and 10 after treatment, fruit peel samples were taken from the original shaded side of the treated fruit and both the sun-exposed side and the shaded side of the control fruit at midday to determine photosynthetic pigments and enzymatic and non-enzymatic antioxidants. Maximum photosystem II efficiency of the original shaded side decreased sharply after 1 day exposure to full sun, and then gradually recovered to a similar value of the sun-exposed side of the control fruit by day 10. The shaded side of the control fruit had much lower xanthophyll cycle pool size and conversion and antioxidant enzymes and soluble antioxidants of the ascorbate-glutathione cycle than the sun-exposed side. In response to full sun exposure, xanthophyll cycle pool size of the original shaded side increased, reaching a similar value of the sun-exposed side by day 10. Ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase and total pool size and reduction state of both ascorbate and glutathione of the original shaded side all increased to the corresponding values found in the sun-exposed side of the control fruit over a 10-day period. It is concluded that both xanthophyll cycle and the ascorbate-glutathione cycle in the original shaded side are up-regulated in response to fullsun exposure to minimize photo-oxidative damage and contributes to its re-acclimation to full sun.